Electrical stimulation unit and waterbath system

ABSTRACT

A method for treating an infected area on a subject, comprising the steps of: exposing the infected area to an aqueous solution; and providing direct current to the aqueous solution to treat the infected area.

TECHNICAL FIELD

The present invention relates generally to the field of treatinginfections, and, more particularly, to an improvedfungicidal/fungistatic treatment system for treating toenail fungus,dermatological fungi, fungal infections, and the like.

BACKGROUND ART

Toenail fungus alone affects 2-13% of the general population of theUnited States; over 30% of the population over 60 years of age areaffected. Current systemic treatment consists of the use of an expensivedrugs or pharmaceutical agents many of which have complications andassociated interactions. These pharmaceutical treatments are less thanideal for many patients because of the cost and danger associated withthem.

Various approaches to treating this disease have been attempted andemployed, and most involve pharmaceutical agents applied topically orsystematically. For instance, U.S. Pat. No. 6,319,957 describes the useof compositions based on glyco-alcohol, hydro-alcohol orglyco-hydro-alcohol solutions of a glycol or glyceric ester of retinoicacid, preferably in association with the ethyl ester of retinoic acidand with hydroquinone, treat unsightly skin disorders such as acne,wrinkles, scars, stretch marks, dark spots, etc., and in treatingmycotic skin diseases and psoriasis.

U.S. Pat. No. 6,303,140 teaches a plaster preparation comprising asynthetic rubber; a reinforcing agent based on silica or randomstyrene-butadiene, copolymer; a tackifier; salicylic acid or apharmaceutically acceptable salt or ester thereof to treat mycoticinfections.

U.S. Pat. No. 6,290,950 describes a new class of mycosis vaccinescomprising homogenised inactivated yeast blastospores and homogenisedinactivated dermatophyte microconidia or antigenic material of saidspores, methods for their production and their use for the prophylaxisand/or treatment of mycoses in mammals, preferably humans. The vaccinesaccording to the present invention are especially useful for theprophylaxis and/or treatment of skin mycosis, preferably dermatomycosisand/or candidosis and/or onychomycosis.

U.S. Pat. No. 6,287,276 describes a set depth nail notcher and methodfor treating nail fungus that is used to cut a notch to a predetermineddepth in a nail or a toe of finger infected with fungus and then apply atopical anti-fungal medication to the toe or finger through the notch.

U.S. Pat. No. 6,281,239 teaches a method of treating onychomycosis byadministering to an infected area around a nail of a patient a tissuesoftening composition containing urea and an antifungal composition inone or separate compositions, concurrently or non-concurrently.

Several studies have reported that electrical stimulation augments woundhealing, Electrical stimulation has been reported to improve blood flow,decrease edema, and inhibit bacterial growth. Numerous studies havereported that monophasic pulsed current from a high voltage pulsedsource (HVPC) augments wound healing. Additional studies have shownsignificant increases in transcutaneous partial pressure of oxygen (tCPO₂) in diabetic individuals following use of electrical stimulation.HVPC has been used to successfully treat diabetic foot ulcers.

Several studies have demonstrated that electrical currents exist inliving organisms. Cells follow the path of this current flow, which isreferred to as the galvanotaxic effect. It is theorized that electricalstimulation augments the endogenous bioelectric system in the body. Theincrease in the rate of wound healing with electrical stimulation isalso theorized to be a result of attraction of different cell types.Studies have shown that migration of macrophages, fibroblasts, mastcells, neutrophils, and epidermal cells is influenced by electricalstimulation. Electrical stimulation has also been shown to increase theproliferation of fibroblasts and protein synthesis, as well as thegrowth of neurites. These factors play a significant role in healing.Furthermore, the tensile strength of the collagen has been shown toincrease upon application of such electrical fields, thus increasing thestrength of the wound scars. For these reasons, the use of electricalstimulation for the treatment of chronic wounds has been usedincreasingly during the last several years.

The term onychomycosis refers to any fungal infection of one or moreelements of the nail system, which consists of the nail matrix, the nailbed and the nail plate. Several studies suggest that onychomycosisaffects between 2% and 18% (or possibly more) of the world's population.In North America, onychomycosis accounts for approximately 50% of allnail disease, is an infection several times more common in the toenailthan the fingernail, and is most commonly found among older individuals.Some studies suggest that nearly 50% of the population over 70 years ofage may be affected. The incidence of onychomycosis in the United Statesand other countries of the developed world has been increasing in recentyears. This is thought to be most likely the result of severalcontributing factors including: the general aging of the population; thepossible higher incidence of diabetes mellitus; the greater use ofimmunosuppressive drugs and antibiotics; the increased exposure of thegeneral population to the etiologic fungi; the HIV epidemic.

Onychomycosis can be caused by three different groups of fungi: thedermatophytes, the yeasts and the nondermatophytic molds. Thedermatophytes are the most common etiology, accounting for between 85%and 90% of all cases. Just two dermatophyte species, Trichophyton rubrum(T. rubrum) and Trichophyton mentagrophytes (T. mentagrophyte), areresponsible themselves for nearly 80% of all cases of onychomycosis.Several different yeast species can also cause onychomycosis. Thesespecies are together responsible for between 5% and 10% all cases. Inapproximately 70% of these cases, the etiological agent is Candidaalbicans. Finally, several different species of the nondermatophytemolds can also cause onychomycosis. As a group, these are responsiblefor approximately 3% to 5% of all cases.

Although onychomycosis is not a fatal infection, and is usually not avery debilitating condition in most afflicted individuals, it can stillhave serious emotional and/or physical consequences. The condition canbe associated with significant pain and discomfort, and in severe cases,it may sometimes lead to disfigurement and/or to various degrees offunctional loss. In addition to physical impairment, the psychologicaland social consequences of onychomycosis can also be significant. Thus,onychomycosis represents far more than a mere cosmetic problem for manyafflicted individuals, and professional treatment from health careproviders is very often sought.

The treatment of onychomycosis, however, has proven difficult. The threetraditional approaches to treatment are debridement of the nail unit,topical medication and systemic chemotherapy. The most successful ofthese approaches has been the use of systemic antifungal drugs. Over thelast 40 years, oral systemic antifungal agents have been the mainstay ofonychomycosis therapy. However, because of several negative factors thatinclude drug toxicity, possible adverse interactions of antifungalagents with other drugs in the body, and the prolonged course oftreatment required with many of these antifungal therapeutic regimes,the search for new, alternative treatments, which are both efficaciousand which present minimal side effects, is still an important researchgoal.

DISCLOSURE OF THE INVENTION

With parenthetical reference to the corresponding parts, portions andsurfaces of the disclosed embodiment, merely for purposes ofillustration and not by way of limitation, the present invention broadlyprovides a method and apparatus for treating infections in human oranimal subjects.

In one aspect, the invention provides a method for treating an infectedarea on a subject, comprising the steps of exposing the infected area toan aqueous solution; and providing direct current to the aqueoussolution to treat the infected area. This method of treatment may alsobe used to treat other infections including onchomycosis, molluscumcontagium, papilloma virus, warts, epidermodysplasia verruciformis,herpes virus, or other fungal infection. The method may be used to treatan infected area wherein the infected area is on the skin of thesubject.

In another aspect of the invention, the aqueous solution includeshydrogen peroxide. Another aspect is where the aqueous solutioncomprises about 0.01 to 3.0 weight percent hydrogen peroxide.

It is an object of the invention to provide direct current of less thanabout 3 milliamperes, or less than about 50 milliamperes. It is anotherobject of the invention to provide direct current supplied by a voltagesource of less than about 150 volts. In another aspect of the inventionthe direct current is pulsed. In another aspect, the direct current hasa pulse width of about 5-50 microseconds. In yet another aspect of thisinvention, the infected area is treated with the direct current for atime period of about 20-45 minutes.

This invention also relates to apparatus for treating an infected areaon a subject comprising: a reservoir; an aqueous solution in thereservoir and exposed to the infected area; a first electrode in thereservoir; a second electrode in the reservoir; and a circuit forproviding current to the aqueous solution to treat the infected area.

In one aspect the infected area is immersed in the aqueous solution.

In another aspect, the first electrode and second electrode are formedof stainless steel.

It is an object of this invention to provide a wearable apparatus forthe treatment an infected area on a subject comprising a membrane madeof a material that is impervious to aqueous solutions and having aperiphery or an edge; an adhesive disposed on the periphery or edge ofthe membrane; an aqueous solution in the membrane in contact with aninfected area of a subject; a first electrode affixed to the membrane; asecond electrode affixed to the membrane; and a circuit for providingcurrent to the aqueous solution to treat the infected area.

It is a further object of the invention to provide such apparatus with aliquid filler opening that allows the apparatus to be placed against theinfected area to form a pocket which is capable of holding an amount ofan aqueous solution.

It is also an object of the invention to provide such apparatus wherethe membrane is adapted to be attached to the subject.

In another aspect of the invention, the first electrode and secondelectrode are formed of stainless steel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a subject's infected area immersed in a reservoir with asource of high or low voltage current available.

FIG. 2 shows an apparatus or apparel that allows for treatment of aninfected area by contacting the infected area with an aqueous solutionproviding a direct current to the aqueous solution across the infectedarea.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Both monophasic pulsed current from a high voltage source (HVPC) anddirect current electrical stimulation from a low voltage source (LVDC)are fungicidal/fungistatic. Humans treated with HVPC electricalstimulation in a waterbath displayed markedly diminished fungalinfection, and sound normal nail growth. The growth of Trichophytonmeiitagrophytes fungi and Trichophyton rubru fungi can be inhibited withdirect current electrical stimulation from a low voltage source. Thefungicidal/fungistatic properties of electrical stimulation on toenailfungus, dermatological fungi, and other fungal infections has beendemonstrated.

EXAMPLE 1

Based on these experiments and the discovery of electrical stimulation'sfungicidal/fungistatic properties, it is logical that a therapeuticdevice could be fashioned consisting of a small electrical stimulationunit and a foot waterbath system. Referring now to the drawings, and,more particularly, to FIG. 1 thereof, an appliance used to treat toenailfungus will comprise a waterbath (1) designed to allow either one orboth feet (2) to fit comfortably and be immersed in solution (3). Anelectrode (4) on either side of the bath will allow a safe current topass through the solution (3) and over and around the toes and thenails. The current will be supplied by an electrical stimulation unit(5), which will be a small device easily attachable to, or built into,the bath (1). The electrical stimulation unit (5) will have leads, whichattach to the electrodes (4). The system used to treat toenail funguswill have leads on either side of the bath, lateral and medial or frontand back. The therapeutic device will be adapted to treating other partsof the body which can be easily immersed, for example, hands.

The device delivers a pulsed current of 0 to 150 volts at peak in pulsepairs of 150-330 microseconds apart. The pulse width is 5-50microseconds and the pair repeat frequency is 100-200 Hz. This device isconnected to the waterbath by way of electrodes, thus allowingelectrical current to travel in the solution and cover the affectedarea. This unit will be as safe as a transcutaneous electrical nervestimulator (TENS) unit presently used for pain modulation, but will bepotentiated to deliver a distinct type of current.

Based on findings of the fungicidal/fungistatic properties of bothpulsed and direct current electrical stimulation, many additionalapplications are possible. In addition to toe nail fungus,dermatological fungi, fungi in wounds and fungal infections areadditional fungi, which could be treated with this system. Also,veterinary applications are possible for animals and livestock withfungal infections are anticipated.

EXAMPLE 2

Referring now to the drawings, and, more particularly, to FIG. 2thereof, the apparatus can be adapted to for wearing or attachment to asubject. The apparatus includes a (1) membrane made of a material thatis impervious to aqueous solutions, filled with an aqueous solution (2)in contact with an infected area of a subject. The edge or periphery ofthe membrane may have an adhesive disposed on said edge of the membraneto provide a seal in order to prevent the aqueous solution from leaking.The apparatus includes two electrodes (3) affixed to the membrane,connected by leads (4) to a circuit (5) for providing current theaqueous solution (2) to treat the infected area. This wearable apparatusmay be a sock,

Apparel like devices can be used to treat the fungal infection byproviding a wearable fluid reservoir to the area to be treated whichalso incorporates a first electrode means and a second electrode means.In some instances, this device could have the general form of a bandageor the like with an adhesive portion along the periphery of thereservoir means to provide a seal with the contact area to be treated.Then, a DC power source can be hooked up to the first and secondelectrode to provide the electronic field across the electrodes and thesurface to be treated. This “bandage” like apparatus allows thetreatment method to be undertaken without limiting the activity of thepatient. A person being treated with such an apparatus will be able tobe active during treatment and will hence be more likely to participatein complete treatment regimens.

Those skilled in the art will recognize that besides patches, alocalized reservoir similar to the “bandage” reservoir means can be madeinto part of a piece of apparel. Depending upon the sites of the fungalinfection the apparel could be in the form of a sock, sweat-pant, orshirt.

EXAMPLE 3

It has also been found that certain additives to the aqueous solution inthe reservoir can increase the efficacy of the treatment regimen. Forinstance, it has been found that an oxygenating source such as hydrogenperoxide accelerates the reduction of onchymycotis. Preferably theconcentration of hydrogen peroxide is in the range of 0.01 to about 2weight percent. The solution at those concentrations can bepre-prepared, or can be freshly prepared just prior to treatment.Solution can also be adjusted for slat concentration so that they areisotonic, and additionally buffer systems can be added so that the pH ofthe solutions remains close to physiological conditions of the tissuebeing treated.

EXAMPLE 4

In order to demonstrate the efficacy of direct current from a lowvoltage source (LVDC) or electrostimulation (E-stim) as an antifungalagent for clinical use in the treatment of onychomycosis pure culturesof Trichophyton rubrum and Trichophyton mentagrophytes grown on a solidagar medium were subjected to clinically relevant doses of LVDC. Todetermine antifungal effects due to germicidal and/or fungistaticactivity of LVDC, the diameters of any zones in the agar around theelectrodes which lacked fungal growth after E-stim were measured andcompared to control cultures.

Zones devoid of fungal growth were observed for both Trichophyton rubrumand Trichophyton mentagrophytes around both the anode and cathode whenclinically relevant doses of LVDC from 500 microamperes to 3milliamperes were applied. In this dose range, LVDC acted fungicidallyin a dose dependent manner.

During the past ten years, new treatment protocols which employelectrical stimulation (E-stim) have begun to be used in the treatmentof certain types of wounds. The clinical efficacy of such treatment isnow well documented. One of the ways in which E-stim probably plays arole in enhancing wound healing is by its antimicrobial effect. Severalin vitro studies have conclusively demonstrated that E-stim applicationis antibacterial to most of the pathogenic bacteria commonly found inwounds. In the course of clinical use of low voltage direct currentelectrostimulation (LVDC E-stim) in wound treatment during the last 10years, it became evident that, in addition to its enhancement of thewould healing process, E-stim also seemed to significantly amelioratemany cases of onychomycosis of the toenail. Subsequently, regularemployment of this modality in the treatment of onychomycosis resultedin clinical success.

In order to begin determining the factors responsible for the observedefficacy of this modality in the treatment of onychomycosis, in vitroexperiments were conducted to evaluate the antifungal effects of LVDCE-stim in the control and/or eradication of the two primary etiologiesof onychomycosis, T. rubrum and T. mentagrophytes. LVDC E-stim isclinically significant in the treatment of onychomycosis in conjunctionwith or alternative to other current therapies.

Materials and Methods

Organisms:

The medium employed to culture all fungi was Sabouraud Dextrose Agar(SDA) in 100 ml petri plates obtained from Becton Dickinson MicrobiologySystems. (Becton Dickinson Microbiology Systems, PO Box 243,Cockeysville, N. Mex. 21030). Pure cultures of the dermatophyte fungi T.rubrum and T. mentagrophytes were obtained from Presque Isle Culturest.Permanent stock cultures of T. rubrum were established by inoculation ofthe organism onto a solid growth medium consisting of SDA in petriplates, and subsequent incubation of these SDA plates at 25° C. for 7days. After this time period, each SDA plate was covered by numerouscolonies of T. rubrum which had coalesced into a homogeneous, fluffyfungal “lawn” (or continuous mass of growth) along the entire surface ofthe agar. Permanent stock cultures of T. mentagrophytes were establishedin the manner described above for T. rubrum. The stock cultures of bothfungi were maintained at 4° C. for the entire time period of theseexperiments and were weekly monitored for viability.

Experimental Procedure and Instrumentation:

All of the LVDC E-stim experiments described in this report wereperformed in a NUAIRE Class 11, Type A/B3 biological safety cabinetusing standard aseptic techniques. All LVDC E-stim was performed using aRich-Mar VI LIDC Stimulator. Independent current readings were made witha BK Precision amp-meter from I Dynascon Corporation.

For each E-stim experiment, a 1.0 cm section of agar containing eitherT. rubrum or T. mentagrophytes growing on the surface was removedaseptically from the respective SDA stock culture plates using a steriledissecting needle. The 1.0 CM2 piece of SDA containing either T. rubrumor T. mentagrophytes was then transferred to 5 ml of sterile 0.9%saline. The sterile saline tube was mixed by vortexing for 10 sec inorder to dislodge the fungal hyphae, conidia and spores from the surfaceof the agar. Using a sterile micropipeter, 250 ml of the fungal-salinesolution were transferred to a new, sterile SDA petri plate. If multipleSDA plates were to be inoculated in a given experiment, this procedurewas performed multiple times from the same fungal-saline solution. Thefungal-saline solution was evenly distributed over the entire surface ofeach of the inoculated SDA plate(s) using a sterile glass spreading rod.The SDA plates were then incubated at 25° C. for 24 hr. At the end ofthis period, a barely visible film of fungal growth covered the entiregrowth medium surface of the SDA plates. Non inoculated control SDAplates were included in each experimental group to insure the sterilityof the medium.

After 24 hr of incubation, electrical current was applied to eachexperimental SDA plate containing either T. rubrum or T. mentagrophytesby a Rich Mar VI LIDC Stimulator. The electrodes used to accomplish thisconsisted of 2 pieces of stainless steel (1 mm diameter and 2.5 cm long)which were permanently inserted through the top portion of a sterilepetri plate 1.9 cm apart from each other, and which were secured withepoxy cement on the outer surface. The electrodes were disinfected andstored in 95% ethanol prior to and between experiments. Just prior toeach experiment, the top portion containing the electrodes was removedfrom the 95% ethanol storage unit, the ethanol was allowed to evaporate,and the electrodes were inserted into the agar of the bottom portion ofa SDA plate containing 24 hr growth of either fungus by closing the topportion over the lower portion of a petri plate. Then, either theelectrodes remained in the agar for 30 min at room temperature (22-24°C.) without LVDC application (0 amperes), or LVDC was applied using theE-stim apparatus described above. A current of either 500 microamperes,1 milliamperes, 2 milliamperes or 3 milliamperes LVDC was applied for 30minutes at room temperature (22-24° C.). All amperages were confirmed bythe use of an independent amp-meter connected in series. In eachexperiment, in order to confirm fungal viability and media soundness, acontrol SDA plate containing the particular fungus used in theexperiment, but which was not subjected to either E-stim or electrodeintrusion, was also included. This plate was inoculated at the sametime, in the same manner and from the same saline tube as theexperimental plates.

After LVDC E-stim, each SDA petri plate was incubated at 25° C. for 24hr to allow for additional fungal growth which could then be easilyvisualized. Following this 24 hr incubation, the diameter of any zoneslacking fungal growth (where no additional fungal growth occurred afterapplication of E-stim) at the location of both the positive and negativeelectrodes was measured to the nearest 0.1 mm using a millimeter rulerand a dissecting microscope. The SDA plates were then incubated for anadditional 3 to 5 days, with additional observations and measurementsmade daily.

To determine if the electrode material itself was toxic to either of thetwo fungi, or if any ethanol (used in disinfection) remained on theelectrodes during the E-stim application (which might kill the fungus orinhibit its growth), SDA plates were inoculated with either T. rubrum orT. mentagrophytes and incubated for 24 hr as described above. Followingthis, the electrodes were inserted into the SDA plate containing eitherof the fungi and allowed to remain for 30 minutes (as previouslydescribed), but no electric current was applied (0 amperes). The SDAplates were then incubated for 7 days as described previously, and thenchecked for the presence of any zones lacking fungal growth during eachof these seven days.

To determine if any toxic or inhibitory antifungal metabolites weregenerated in the SDA growth medium as a result of the application ofelectric current, LVDC of either 3 or 8 milliamperes was applied to aSDA plates (as described) for 30 min prior to inoculation of either T.rubrum or T. mentagrophytes. Immediately following this, 250 microlitreof fungal-saline solution of either T. rubrum or T. mentagrophytes wasevenly distributed overeat surface as described previously. The SDAplates were then incubated at 25° C. for 7 days, and the presence of anyzones lacking fungal growth near the site of the electrode contacts (orelsewhere) was observed and/or measured each day.

To determine if LVDC E-stim is acting primarily in a fungistatic manner(inhibited fungal growth but did not kill fungal cells) or a fungicidalmanner (killed fungal cells), the following was done during eachexperiment with either T. rubrum or T. mentagrophytes. LVDC E-stim wasapplied as described above, and after 24 hr incubation at 25° C.,samplings were carefully taken in the areas lacking fungal growth aroundeach electrode with a sterile swab in order to determine if viablefungal cells were present in these zones. This swab was then used toinoculate fresh, sterile SDA plates. These plates were incubated for 7days at 25° C. and daily observed for the presence of any fungal growththat would result from the transfer of any viable fungal hyphae orspores from an experimental plate to the new SDA plate. A control plateinoculated with fungi taken from the same experimental plate, but from aregion away from the electrodes and containing fungal growth, was alsoincluded with each of these experiments.

Results:

In this investigation, several clinically relevant doses of LVDC E-stimwere applied to 24 hr pure cultures of T. rubrum or T. mentagrophytesgrowing on SDA plates. Following this, the diameter of any zones aroundeach electrode lacking fungal growth were observed and measured. Foreach fungus, each experiment was replicated three times at the specifiedcurrent and time settings. The size of the zones around each electrodethat lacked fungal growth for T. rubrum or T mentagrophyte due to theapplication of LVDC E-stim at the various amperages used. For allamperages except 0 amperes, circular or roughly circular zones devoid offungal growth were observed around both the positive (anode) andnegative (cathode) electrodes. In addition, we observed an increase inthe diameter of the zones with increasing amperages for both fungi (FIG.1). Because we inserted 1 mm electrodes into the semi-solid agar surfaceduring each experiment in order to generate a current, there was adepression produced in the agar surface at the location of theseelectrodes on all SDA plates. This was due, most likely, to compressionand/or liquefaction of the agar. Consequently, the absence of growthobserved directly under each electrode in this study, as noted in aprevious was not considered to be indicative of fungicidal orfungistatic activity.

In order to confirm that the antifungal effects on T. rubrum and T.mentagrophytes observed in this study were due to the application ofLVDC E-stim and not to other possible causes, the following controlswere done. To rule out the possibility that the observed antifungaleffects were due to the electrode material itself, in each round ofexperiments, a SDA plate was inoculated with either fungus and incubatedfor 24 hr. After this, the electrodes were inserted in the same mannerand for the same time period as we would in a typical experiment usingLVDC, but no current was applied (0 amperage). The plates were thenincubated and observed as described above. On these SDA plates, no areasdevoid of fungal growth around either electrode were ever observed witheither T. rubrum or T. mentagrophytes. This same experiment was alsoused to determine if the lack of fungal growth around the two electrodeswas due to any remaining ethanol (used to disinfect the electrodesbetween experiments) on the electrodes. If this were the case, some areadevoid of fungal growth should have been observed around the regionwhere the electrodes were inserted into the agar plates, even withoutthe application of LVDC (0 amperes). No such region was observed.

Another possible reason for the antifungal effects observed in thisinvestigation was that changes produced in the fungal growth medium(SDA) as a result of LVDC E-stim application made the medium no longersupportive of fungal growth. To investigate this possibility we did thefollowing set of experiments. Prior to the inoculation of either fungusonto the SDA medium, LVDC of either 3 milliamperes (the highest clinicalamperage used in this study) or 8 milliamperes (beyond the clinicalrange used) was applied to 8 SDA plates for 30 min each (4 plates at 3milliamperes, and 4 plates at 8 milliamperes). T. rubrum was theninoculated on 2 of the 3 milliampere and 2 of the 8 milliampere platesand incubated for 7 days. T. mentagrophytes was inoculated similarly. Ifthe medium was indeed changed by LVDC E-stim application in such a wayas to prevent or inhibit fungal growth, this should be observed as alack of growth in the agar in the region around the electrodes (orpossibly some other region of the agar). No such region lacking fungalgrowth was observed for either T. rubrum or T. mentagrophytes in theareas surrounding either of the electrodes (or any other area) when weinoculated the fungi onto a SDA plate after 3 milliamperes of LVDCapplication. At 8 milliamperes, no region lacking fungal growth wasobserved for either fungus at the cathode. However, at this amperage,some discoloration, liquefaction and subsequent depression of the agarwas observed in the area in which the anode was placed. Neither funguswas able to grow very well over this discolored, depressed anode regionat 8 milliamperes. Otherwise, growth occurred throughout the remainderof the plate.

In order to determine if the range of LVDC E-stim application used inthese experiments was acting primarily as an fungicidal agent orfungistatic agent, the areas around each electrode that lacked fungalgrowth were sampled with a sterile swab 24 hr after E-stim applicationfor the presence of viable fungal cells or spores. A total of 24samplings for T. rubrum and 24 samplings for T. mentagrophytes weremade. A sampling was taken from the area around each electrode on allplates which received a dose of 500 microamperes, 1 milliampere, 2milliamperes and 3 milliamperes. Growth was observed on the fresh SDAplates inoculated with a swab after sampling an experimental plate. Inthe remaining 22 samplings, no growth was observed.

Finally, as has previously been reported, the production of gas bubblesat the cathode during all experiments, and occasionally, also at theanode was observed. In addition, a blue discoloration was observedaround the cathode, possible due to a pH change.

Discussion:

This investigation was undertaken in order to determine if theclinically observed efficacy of LVDC E-stim in the treatment ofonychomycosis is due (at least in part) to an antifungal effect of themodality. Antibacterial effects against many of the common bacterialwound pathogens have been demonstrated in vitro for both low voltagedirect current and high voltage pulsed current in several studies.However, such in vitro documentation is negligible with respect to theeffect of electric current on the common fungal pathogens, the yeastCandida albicans being one of the few exceptions. Clinically relevantdoses of LVDC E-stim are antifungal in vitro to the two primary causesof onychomycosis, the fungi T. rubrum and T. mentagrophytes. Theantifungal effects of LVDC E-stim occur in a dose dependent manner inthe clinically relevant ranges used (500 microamperes to 3 milliamperes)in these in vitro experiments.

A second and related determination to be made was whether LVDC E-stim inthe dose range used was acting primarily as a fungistatic agents or afungicidal agent. To determine this, the areas devoid of fungal growtharound the electrodes in each experiment were carefully sampled with asterile swab 24 hr after E-stim to assay for any viable fungal cells orspores which could give rise to fungal colonies on new SDA plates.Similar methodologies have been used to determine if E-stim isbactericidal or bacteriostatic. In 46 of the total of 48 samplings, nofungal growth was observed on the newly inoculated SDA plates. The twoplates that did show some growth were both from the cathode region ofthe 3 milliampere dose plate. These zones had the largest diameter, andit is possible that the sampling swab may have inadvertently touchedsome viable fungal cells at the periphery of the zone. As such, theywould represent an artifact of these experiments, rather than actuallack of fungicidal activity, which is likely the case. Consequently, thedata strongly suggest that LVDC E-stim is acting fungicidally in theamperage range used in this study.

Cellular death can be brought about by a number of factors that include:damage or denaturation of key cellular enzymes; damage to DNA; damage ordisruption of the cell membrane; damage or destruction of key cellulartransport systems. Electricity is believed to most likely kill cells byaffecting the molecular structure of the cell membrane, leading to fatalchanges in cell membrane permeability. Such cell membrane damage couldexplain the antifungal effects of LVDC E-stim observed both in vivo andin vitro. However, other factors might also play a role. Application ofelectric current to the agar medium can result in changes in the pH ofthe medium, increases in temperature and the generation of toxicmetabolites. All of these (and possibly others still) could actantimicrobially to one degree or another. Such factors have beenconsidered in other studies looking at the antibacterial effects ofelectricity. These factors were not individually examined in this studywhich was mainly concerned with determining if LVDC E-stim applicationitself was antifungal. If the application of electricity to a fungusgrowing on a toenail or agar surface results in the death of all or someof those fungal cells, then whether that fungicidal activity was due tofatal changes in the cell membrane of fungal cells and also to changesin pH or increases in cellular temperature is secondary to the presentinterest. Such aspects will hopefully be addressed in subsequentinvestigations.

However, this present investigation sought to demonstrate that anyantifungal effects of LVDC E-stim on the two major etiologies ofonychomycosis were due to the application of E-stim itself (due to theeffects of electrical current), and not due to any artifacts produced asa result of our experimental methodology. Because no fungal inhibitionwas observed around either electrode during any trial when theelectrodes were inserted but when current was not applied (0 amperes),the electrode material itself (stainless steel) is not likelyantifungal. Another concern was that some of the ethanol, used todisinfect the electrodes before and in between experiments, might remainon the electrodes upon insertion into the SDA medium. Since ethanol is adisinfectant, it can kill and or inhibit fungal and bacterial cells.Thus, areas around each electrode lacking fungal growth may be due tothe disinfection action of ethanol, rather than the antifungal effectsof the LVDC E-stim. To minimize the possibility of this, after removalof the electrodes from their ethanol storage unit (stored in abiological hood), they were allowed to dry for a minimum of 30 secondsbefore they were inserted into any SDA experimental plates. Noanti-fungal activity was observed at the 0 amperage setting as describedabove, and insufficient (or no) ethanol remained on the electrodes uponinsertion into any SDA plate. It is clear that all the ethanol hadevaporated and was not a cause of the observed zones lacking fungalgrowth.

Another possible reason for any observed areas lacking fungal growtharound either electrode was that the application of LVDC E-stim in thecurrent range changed the SDA medium in some way so that is could nolonger support the growth of either T. rubrum or T. mentagrophytes. Thiscould result from the production of toxic and or inhibitory products inthe medium as a result of the LVDC application, or from changes in thepH of the medium due to LVDC, or from the denaturation and degenerationof necessary nutrients in the medium without which the fungi could notgrow. To investigate this possibility, either 3 milliamperes or 8milliamperes of LVDC E-stim was applied to several SDA plates beforethey were inoculated with either T. rubrum or T. mentagrophytes. If theSDA medium was indeed changed by LVDC application so that it nowprevented or inhibited fungal growth, this should be observed as a lackof growth in/on the agar in the region(s) around the electrodes (orpossibly some other region of the agar). As the data show, no suchregion was observed at 3 milliamperes around either the cathode or theanode, or anywhere else on the plates. Growth occurred throughout eachSDA plate. At 8 milliamperes, more than twice the highest amperage usedin this study, growth occurred at the cathode, but not the anode, whereliquefaction and depression was observed. It is probable that at thisamperage, lack of growth was due to some physical changes produced inthe medium (i.e., liquefaction), or to the generation of toxicmetabolites, or the denaturation of vital nutrients, or a combination ofseveral factors. However, because 3 milliamperes was the highest dose ofLVDC E-stim used in these antifungal studies, at this amperage and atthe lower amperages, the media was not altered in a significant way soas to negatively affect the growth of either fungus. To further supportthis assertion, in the experiments involving 500 microamperes, 1milliampere, 2 milliamperes and 3 milliamperes of LVDC E-stim, the areasof the agar lacking fungal growth due to the LVDC application were stillcapable of supporting the growth of both fungi. This is evidenced by thefact that if these agar plates were incubated at 25° C. for 7 to 10days, eventually viable fungi from outside the diameter of thefungicidal zone would clearly begin to re-colonize that zone until asolid fungal lawn was again formed over the entire area. Together, theseresults strongly support the assertion that any possible changesproduced in the SDA medium were not responsible for the observed zoneslacking fungal growth around the electrodes in the clinically relevantdose range of LVDC that we employed in this study.

EXAMPLE 5

Similar to LVDC discussed in Example 4, monophasic pulsed current from ahigh voltage pulsed source(HVPC) may also be used as another preferredembodiment of this invention. For HVPC, the direct current is suppliedby a voltage source of less than about 150 volts.

Therefore, while several preferred forms of the inventive method andapparatus have been shown and described, and several modificationsthereof discussed, persons skilled in this art will readily appreciatethat various additional changes and modifications may be made withoutdeparting from the spirit of the invention, as defined anddifferentiated by the following claims.

1. A method for treating an infected area on a subject, comprising thesteps of: exposing said infected area to an aqueous solution; andproviding direct current to said aqueous solution to treat said infectedarea.
 2. The method set forth in claim 1 wherein said area is infectedwith one of onchomycosis, molluscum contagium, papilloma virus, warts,epidermodysplasia verruciformis, herpes virus, or fungal infection. 3.The method set forth in claim 1 wherein said infected area is on theskin of said subject.
 4. The method set forth in claim 1 wherein saidaqueous solution includes hydrogen peroxide.
 5. The method set forth inclaim 4 wherein said aqueous solution comprises about 0.01 to about 3.0weight percent hydrogen peroxide.
 6. The method set forth in claim 1wherein said direct current is less than about 3 milliamperes.
 7. Themethod set forth in claim 1 wherein said direct current is less thanabout 50 milliamperes.
 8. The method set forth in claim 1 wherein saiddirect current is supplied by a voltage source of less than about 150volts.
 9. The method set forth in claim 1 wherein said direct current ispulsed.
 10. The method set forth in claim 9 wherein said direct currenthas a pulse width of about 5-50 microseconds.
 11. The method set forthin claim 1 wherein said infected area is treated with said directcurrent for a time period of about 20-45 minutes.
 12. An apparatus fortreating an infected area on a subject comprising: a reservoir; anaqueous solution in said reservoir and exposed to said infected area; afirst electrode in said reservoir; a second electrode in said reservoir;and a circuit for providing current to said aqueous solution to treatsaid infected area.
 13. The apparatus set forth in claim 12 wherein saidinfected area is immersed in the aqueous solution.
 14. The apparatus setforth in claim 12 wherein said first electrode and second electrode areformed of stainless steel.
 15. A wearable apparatus for the treatment aninfected area on a subject comprising: a membrane made of a materialthat is impervious to aqueous solutions and having an edge; an adhesivedisposed on said edge of said membrane; an aqueous solution in saidmembrane in contact with an infected area of a subject; a firstelectrode affixed to said membrane; a second electrode affixed to saidmembrane; a circuit for providing current to said aqueous solution totreat said infected area.
 16. The apparatus set forth in claim 15further comprising a liquid filler opening that allows said apparatus tobe placed against the infected area to form a pocket which is capable ofholding an amount of an aqueous solution.
 17. The apparatus set forth inclaim 15 wherein the membrane is adapted to be attached to said subject.18. The apparatus set forth in claim 15 wherein said first electrode andsecond electrode are formed of stainless steel.